Structural and functional characterization of human Iba proteins Jo ¨ rg O. Schulze 1 , Claudia Quedenau 2 , Yvette Roske 1 , Thomas Adam 3 , Herwig Schu ¨ ler 1 , Joachim Behlke 1 , Andrew P. Turnbull 1 , Volker Sievert 2 , Christoph Scheich 2 , Uwe Mueller 4 , Udo Heinemann 1,5 and Konrad Bu ¨ ssow 2,6 1 Max Delbru ¨ ck Center for Molecular Medicine, Berlin, Germany 2 Max Planck Institute for Molecular Genetics, Berlin, Germany 3 Institute of Microbiology and Hygiene, Charite ´ Medical School, Berlin, Germany 4 Macromolecular Crystallography, BESSY GmbH, Berlin, Germany 5 Institute of Chemistry and Biochemistry – Crystallography, Free University, Berlin, Germany 6 Department of Structural Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany Iba1, also known as allograft inflammatory factor 1 (AIF-1), is a 17-kDa protein with a central pair of EF-hand motifs [1]. This feature is common to a large family of Ca 2+ -binding proteins known as EF-hand proteins [2]. Iba1 was found to bind calcium ions in overlay assays [3]. The structure of human Iba1 (h-Iba1) was determined by X-ray crystallography (PDB code 2d58) [4] and NMR (unpublished; PDB code 2G2B). Both techniques revealed a monomeric, Ca 2+ -free protein. However, the crystal structure of Iba1 from mouse (m-Iba1; PDB code 1wy9) showed a homodimeric protein with Ca 2+ bound to only the second EF-hand motif [4]. Thus, the dimerization of Iba1 was suggested to be induced by Ca 2+ binding. A homolog of Iba1 named C9orf58 or Iba2 was revealed by the Human Genome Project. Human Iba2 consists of 150 amino acids (17 kDa), and the sequence identity to h-Iba1 is 60%. A systematic microarray Keywords actin cross-linking; allograft inflammatory factor 1; calcium binding; EF-hand; ionized calcium binding adapter molecule Correspondence U. Heinemann, MDC, Robert-Ro ¨ ssle-Str. 10, 13125 Berlin, Germany Fax: +49 30 9406 2548 Tel: +49 30 9406 3420 E-mail: heinemann@mdc-berlin.de Database The coordinates of both structures have been deposited in the RCSB Protein Data Bank under PDB codes 2vtg and 2jjz (Received 20 March 2008, revised 18 July 2008, accepted 22 July 2008) doi:10.1111/j.1742-4658.2008.06605.x Iba2 is a homolog of ionized calcium-binding adapter molecule 1 (Iba1), a 17-kDa protein that binds and cross-links filamentous actin (F-actin) and localizes to membrane ruffles and phagocytic cups. Here, we present the crystal structure of human Iba2 and its homodimerization properties, F-actin cross-linking activity, cellular localization and recruitment upon bacterial invasion in comparison with Iba1. The Iba2 structure comprises two central EF-hand motifs lacking bound Ca 2+ . Iba2 crystallized as a homodimer stabilized by a disulfide bridge and zinc ions. Analytical ultra- centrifugation revealed a different mode of dimerization under reducing conditions that was independent of Ca 2+ . Furthermore, no binding of Ca 2+ up to 0.1 mm was detected by equilibrium dialysis. Correspondingly, Iba EF-hand motifs lack residues essential for strong Ca 2+ coordination. Sedimentation experiments and microscopy detected pronounced, indistin- guishable F-actin binding and cross-linking activity of Iba1 and Iba2 with induction of F-actin bundles. Fluorescent Iba fusion proteins were expressed in HeLa cells and co-localized with F-actin. Iba1 was recruited into cellular projections to a larger extent than Iba2. Additionally, we stud- ied Iba recruitment in a Shigella invasion model that induces cytoskeletal rearrangements. Both proteins were recruited into the bacterial invasion zone and Iba1 was again concentrated slightly higher in the cellular extensions. Abbreviations AIF-1, allograft inflammatory factor 1; CFP, cyan fluorescent protein; Iba, ionized calcium binding adapter molecule; TEV, tobacco etch virus; YFP, yellow fluorescent protein. FEBS Journal 275 (2008) 4627–4640 ª 2008 The Authors Journal compilation ª 2008 FEBS 4627 study has revealed expression profiles for most of the human transcripts and uncovered different tissue- specific expression of Iba1 and Iba2 [5]. For Iba1, pref- erential expression in spleen, tonsil, lymph node, thymus and also lung was found, confirming previous results [6,7]. Pronounced expression in the kidney was found for Iba2. Iba1 is upregulated in mononuclear cells found in transplanted hearts during the course of allograft rejection [7,8] and in heart arteries injured by balloon angioplasty [6]. Iba1 expression in vascular smooth muscle cells is induced upon tissue injury by cytokines [9]. Iba1 expression was examined in mouse using comprehensive immunohistochemistry. All sub- populations of macrophages were positive, except for alveolar macrophages [10]. Spermatids were the only cells not belonging to the monocyte ⁄ macrophage line- age expressing Iba1 [10]. Organized actin cytoskeleton remodeling is essential for macrophages and Iba1 was found to bind and cross-link filamentous actin (F-actin) [11,12] and to translocate to lamellipodia, membrane ruffles and phagocytic cups [3,12]. Iba1 cooperates with L-fimbrin, another F-actin-bundling protein, as it was shown to directly bind fimbrin and to enhance its activity [13]. This study is the result of a systematic analysis of proteins [14] encoded by clones from the German cDNA Consortium [15]. LIFEdb, a database integrat- ing systematic studies with this cDNA collection, includes information on the subcellular localization of the corresponding proteins [16,17]. LIFEdb reports co-localization with the cytoskeleton and adhesion plaques for the Iba2 cDNA clone DKFZp761J191 derived protein. The structure presented here reveals functional simi- larities and differences between Iba1 and Iba2. We investigated Ca 2+ binding and homodimerization of Iba1 and Iba2. Furthermore, F-actin binding and cross-linking assays were performed with both human Iba proteins, and their role in bacterial invasion was investigated. Results Crystal structures of Iba2 Human Iba2 crystallized into two different forms under the same conditions. The first form (Iba2 t ) grew in the trigonal space group P3 2 21 and contained one molecule per asymmetric unit. The second form (Iba2 o ) crystallized in the orthorhombic space group P2 1 2 1 2 with four molecules in the asymmetric unit (Table 1). The Iba2 structure was solved by molecular replace- ment using m-Iba1 (PDB code 1WY9) [4] as a search model, which shares a sequence identity of 60% with Iba2. The crystal structure of Iba2 t was refined to a maximal resolution of 2.45 A ˚ , whereas Iba2 o was refined to 2.15 A ˚ . Iba2 is a compact, single-domain protein composed mainly of a helices (Figs 1 and 2). The core of Iba2 is a pair of EF-hand motifs, denoted as EF-hands 1 and 2, each consisting of two a helices (aA, aB and aC, aD, respectively) flanking a loop region able to bind calcium ions in EF-hand proteins [18]. As commonly observed in EF-hand proteins [18], the two motifs have an approxi- mately twofold rotation symmetry with a pseudo-dyad axis passing through the small anti-parallel b sheet in the center. Despite extensive efforts, no crystal structure with calcium ions bound to the EF-hands could be obtained. Two additional helices (aN and aE) comple- ment the EF-hand pair on both termini. Twelve residues at the N-terminus and 23 at the C-terminus are not visible in the final electron density maps of the five Iba2 molecules observed in total. Thus, the termini are either flexible or degraded. All five molecules show basically the same conformation with rmsd values of Ca atoms ranging from 0.77 to 0.91 A ˚ . In one molecule of Iba2 o , Table 1. Data collection and refinement statistics. The values in parentheses refer to the shell of highest resolution. Crystal form Iba2 t Iba2 o Data collection Unit cell dimensions, a, b, c (A ˚ ) 70.5, 70.5, 95.2 71.5, 186.5, 51.4 Space group P3 2 21 P2 1 2 1 2 Wavelength (A ˚ ) 0.9184 0.9184 Number of unique reflections 10 457 38 251 Resolution range (A ˚ ) 30–2.45 (2.58–2.45) 30–2.15 (2.25–2.15) Completeness of data (%) 99.6 (99.9) 99.7 (100) Redundancy 7.9 (8.0) 7.3 (7.4) R sym (%) 6.0 (64.8) 8.2 (75.3) <I ⁄ r(I)> 20.5 (3.5) 16.1 (3.0) Refinement Maximal resolution (A ˚ ) 2.45 (2.51–2.45) 2.15 (2.21–2.15) No. of atoms: protein, water 896, 40 3406, 221 Monomers per asymmetric unit 14 R-factor (%) 20.4 (34.1) 21.6 (26.5) R free (%) 23.2 (24.9) 25.7 (32.3) Average B-factor (A ˚ 2 ) 36.6 38.1 rmsd bond length (A ˚ ) 0.016 0.017 rmsd bond angles (°) 1.7 1.6 Ramachandran plot a 95.0 ⁄ 5.0 ⁄ 0 ⁄ 0 94.8 ⁄ 5.2 ⁄ 0 ⁄ 0 a PROCHECK [45]: most favored ⁄ additionally allowed ⁄ generously allowed ⁄ disallowed region. Human Iba proteins J. O. Schulze et al. 4628 FEBS Journal 275 (2008) 4627–4640 ª 2008 The Authors Journal compilation ª 2008 FEBS however, residues 74–90 of the helix–loop–helix region aB–aC are not resolved. The overall topology of Iba2 shows structural simi- larities to classical EF-hand proteins. The second pair of EF-hands in calmodulin (PDB code 1CLL) [19] and the second pair of EF-hands in troponin C (PDB code 1NCX) [20] are structurally closely related to Iba2. The corresponding DALI Z-scores are 8.2 and 7.2, respectively. As expected, h-Iba1 [4] is structurally most closely related to Iba2 (Z-score 13.4). Unusual dimerization of Iba2 In crystal form Iba2 t , which contains one monomer per asymmetric unit, a homodimer is assembled that contains a central disulfide bridge between Cys35 resi- dues of adjacent molecules related by crystallographic symmetry along a twofold rotation axis (Fig. 2). This dimer is stabilized further by the coordination of two Zn 2+ ions by Glu28 and Glu43 side chains in the dimerization interface. Zn 2+ was provided by the crystallization solution, which contained 100 mm zinc acetate. Nevertheless, the dimerization interface is relatively small and includes only three hydrogen bonds between the dimer subunits. The interface com- prises $ 360 A ˚ 2 corresponding to only 5% of the total solvent accessible surface area of one Iba2 molecule. Crystal form Iba2 o with its four molecules in the asymmetric unit contains two similar homodimers, which are formed by non-crystallographic symmetry in this case. These two dimers feature identical Cys35– Cys35¢ disulfide bridges as observed in crystal form Iba2 t . Moreover, there is only one Zn 2+ ion bound in each dimerization interface of Iba2 o and the Zn 2+ ions are additionally coordinated by His85 side chains of adjacent molecules. There are two additional Zn 2+ ions bound by the Glu64 and Asp107 side chains of adjacent molecules as well as by the Glu32 side chains of neighboring molecules along a twofold rotation axis. These ions form stabilizing crystal contacts, but are not located in potential dimerization or oligomeri- zation interfaces. Dimerization in solution Purification of Iba2 was always performed in the presence of a reducing agent, dithiothreitol. SDS ⁄ PAGE without prior reduction confirmed the Fig. 1. Sequence alignment of human Iba1 [4] and Iba2. Identical residues are shown in black, non-identical residues in gray. Assigned sec- ondary structure elements are depicted in green for Iba1 and in blue for Iba2. The terminal regions of both proteins are not resolved. The EF-hand motifs are framed in red; a consensus EF-hand [26] is shown for comparison. The residues involved in Ca 2+ binding are highlighted in orange. Fig. 2. Cartoon representation of the homodimer in crystal form Iba2 t . One subunit of the dimer is rendered in gray, the other subunit is shown in blue with a trans- parent surface. Important residues in the dimerization interface are depicted in stick representation with oxygen atoms in red and sulfur atoms in yellow. The figures were produced using PYMOL [46]. J. O. Schulze et al. Human Iba proteins FEBS Journal 275 (2008) 4627–4640 ª 2008 The Authors Journal compilation ª 2008 FEBS 4629 absence of disulfide bonds in purified Iba2 (data not shown). However, formation of a disulfide-bridged dimer by oxidation was observed upon removal of dithiothreitol and incubation at room temperature. It is likely that the disulfide bond in the Iba2 structures formed during crystallization after the dithiothreitol in the buffer had been oxidized. Analytical ultracentrifu- gation showed that human Iba1 and Iba2 form homodimers under reducing conditions with dissocia- tion constants of $ 150 and 20 lm, respectively (Fig. 3). The presence of Ca 2+ had only a marginal effect on the dimerization of both Iba proteins. F-actin binding and cross-linking Iba1 is known to bind and cross-link actin polymers [12]. We found that both Iba1 and Iba2 co-sedimented to a similar extent with actin polymers in ultracentrifu- gation experiments (Fig. 4A). Removal of Ca 2+ by EGTA had no effect on the co-sedimentation of Iba1 and Iba2. Actin polymers alone do not sediment during centri- fugation at 8000 g (Fig. 4B). When added at a 0.1 : 1 molar ratio, both Iba1 and Iba2 efficiently shifted F-actin into low-speed pellets, indicating extensive F-actin cross-linking. This effect was even stronger at higher molar ratios. Ca 2+ dependence of the cross- linking activity was not tested. Actin polymers specifically stained with a fluores- cent phalloidin analog appear as a loose network of thin fibers with occasional formation of bundles (Fig. 5A). Bundle formation differs between actin iso- forms and is a function of polymer concentration and ionic strength [21]. At a 0.1 : 1 molar ratio, both Iba proteins completely abolish the background of thin actin fibers and cross-link all actin polymers into bundles (Fig. 5B,C). There are no apparent differences Fig. 3. Homodimerization of Iba1 and Iba2. The molecular mass of Iba proteins against protein concentration was determined by ana- lytical ultracentrifugation in the presence or absence of calcium ions. Fig. 4. F-actin co-sedimentation and cross-linking. (A) Increasing amounts of Iba1 and Iba2 (0–4 l M) were incubated with 4 lM F-actin in the presence of Ca 2+ or EGTA. Proteins were sedimented by ultracentrifugation and pellets analyzed by SDS ⁄ PAGE and Coo- massie Brilliant Blue staining. (B) F-actin alone or mixed with Iba1 or Iba2 was subjected to low-speed centrifugation (8000 g). SDS ⁄ PAGE of pellets (p) and supernatants (s) shows that F-actin sediments readily in the presence of Iba1 and Iba2. Human Iba proteins J. O. Schulze et al. 4630 FEBS Journal 275 (2008) 4627–4640 ª 2008 The Authors Journal compilation ª 2008 FEBS in the filament cross-linking efficiency of Iba1 and Iba2 or in the overall morphology of the generated filament bundles. Calcium affinity of Iba1 and Iba2 Homodimerization and actin binding of Iba1 and Iba2 were similar in the absence or presence of calcium ions. Calcium binding in solution was assayed by equi- librium dialysis. Iba1, Iba2 and calmodulin as positive control were dialyzed against a CaCl 2 solution that was labeled using a trace amount of radioactive 45 CaCl 2 . When protein samples are subjected to equilibrium dialysis, they end up with the same concentration of free ligand molecules as in the dialysis buffer and with additional ligand molecules bound to the protein. An increased Ca 2+ concentration was observed in the dia- lyzed calmodulin sample due to binding of Ca 2+ (Fig. 6). No calcium binding was observed for Iba1 and Iba2. Cellular localization and recruitment to sites of Shigella invasion We expressed cyan fluorescent protein (CFP)-tagged Iba2 in HeLa cells and found that the construct co-localized with F-actin (Fig. 7A–C), in particular with subcortical filaments. Iba2 was also found in cellular projections and adhesion structures, but it was less concentrated in these structures. Recruitment of yellow fluorescent protein (YFP)-tagged Iba1 into cell adhesion plaques and cellular projections was more pronounced in comparison (Fig. 7D–F). In order to verify these potential differences in recruitment patterns of Iba isoforms, we studied Iba recruitment in a Shigella invasion model known to induce major cytoskeletal rearrangements [22]. Here, we show that both Iba proteins are recruited into the bacterial invasion zone. Again, Iba2 seemed to be less concentrated in membrane ruffle-like cellular protrusions (Fig. 8A–D) compared with Iba1 (Fig. 8E–H). In order to verify this relatively subtle difference in protein recruitment behavior between the two Iba proteins, we studied both Iba1 and Iba2 constructs in individual cells. We therefore double- transfected HeLa cells with both Iba constructs, infected the cells and obtained Iba1- or Iba2-specific recruitment patterns in individual cells. As shown in Fig. 8I–L, Shigella-induced Iba2 recruitment into cellular protrusions was less pronounced than the Iba1 pattern. Discussion Structural comparison of Iba2 with Iba1 Structurally, the Iba2 monomer is very similar to monomeric h-Iba1 [4] (Fig. 9A). The rmsd value of common C a atom positions is 1.5 A ˚ . The two struc- tures differ significantly only in the conformation of EF-hand 2 (as discussed below). Fig. 5. Fluorescence microscopy of actin cross-linked by Iba1 and Iba2. (A) Actin polymers stained with a fluorescent phalloidin analog. (B,C) Actin polymers in the presence of Iba1 (B) and Iba2 (C). (D,E) For comparison, actin polymers are shown in the presence of the actin cross- linking protein neurabin-2 (D) and an actin binding deficient truncation mutant of neurabin-2 (E). Fig. 6. Calcium-binding assay for Iba1 and Iba2. Iba1, Iba2 and calmodulin where dialyzed against 100 l M CaCl 2 and a trace amount of 45 CaCl 2 . Upon dialysis, an increased Ca 2+ concentration was found in the calmodulin sample. J. O. Schulze et al. Human Iba proteins FEBS Journal 275 (2008) 4627–4640 ª 2008 The Authors Journal compilation ª 2008 FEBS 4631 Dimeric m-Iba1 [4], by contrast, deviates more from Iba2 as substantiated by an rmsd of 4.2 A ˚ . There is an overall conformational rearrangement including a posi- tional shift of helices aB and aC (Fig. 9B). But most notably, the C-terminal helix aE is relocated by up to 15 A ˚ for residue Ile117 of m-Iba1 (black arrow in Fig. 9B). This dramatic rearrangement opens the dimerization interface and enables the tight interaction of the subunits in the m-Iba1 homodimer. It was sug- gested that the movement of aE is induced by Ca 2+ binding to EF-hand 2 [4]. The three crystal structures have in common that the terminal residues of the Iba proteins are not visible in the electron density. Residues 12–16 of the N-termi- nus and 20–23 of the C-terminus are missing in these structures. For the Iba1 crystal structures, this obser- vation was attributed to a partial truncation of the proteins. Furthermore, an NMR structure of h-Iba1 (PDB code 2G2B) verifies that 17 N-terminal and 18 C-terminal residues are indeed unstructured. Iba2 is a homodimer in the crystal but not in solution The homodimer of Iba2 observed in both crystal forms has a dimerization interface of 360 A ˚ 2 corresponding to 5% of the total protein surface. This interface is unusually small for physiological homodimers because $ 1000 A ˚ 2 would be expected for a 17-kDa protein on average [23]. The Iba2 homodimer contains a central disulfide bond formed by Cys35 residues of both subunits. Con- sidering that the crystallization of Iba2 is difficult to reproduce, disulfide bond formation seems to occur after consumption of the reducing agent dithiothreitol by oxygen in the crystallization plates. Residue Cys35 is not conserved in Iba1. In Iba2, it appears to be con- served, but it must be noted that its gene has only been sequenced from four mammals so far. Furthermore, the dimer in Iba2 t contains two Zn 2+ ions bound in a symmetrical fashion inside the inter- face, whereas both dimers in Iba2 o coordinate only one Zn 2+ ion each in an asymmetric geometry. This inconsistency suggests that the Zn 2+ coordination may not appear in vivo, but only during crystallization in the presence of 100 mm zinc acetate, where it provides essential crystal contacts. In conclusion, the homodi- merization observed in both Iba2 structures seems to be restricted to the crystalline state. It was shown previously that symmetric proteins, such as homodimers, crystallize more readily on average than asymmetric, monomeric proteins by a factor of $ 1.5 [24]. Thus, it was suggested to dimer- ize monomers artificially by disulfide bonds between single cysteine residues introduced by site-directed mutagenesis [24]. In the case of Iba2, a natural cys- teine residue causes the protein to dimerize. Further- more, the Zn 2+ -coordination contacts prevent the dimer from rotating around the disulfide bond. In crystal form Iba2 t , the internal symmetry of the Fig. 7. Iba proteins co-localize with F-actin in HeLa cells. HeLa cells were transfected with YFP-tagged human Iba1 (B) or CFP- tagged Iba2 (E). F-actin was stained using Alexa-594–phallacidine (C,F). Overlay images (A,D) show co-localization of Iba1 and Iba2 with subcortical F-actin. Iba2 is recruited to a lesser extent into cellular projections and adhesion structures than Iba1. Human Iba proteins J. O. Schulze et al. 4632 FEBS Journal 275 (2008) 4627–4640 ª 2008 The Authors Journal compilation ª 2008 FEBS dimer is embodied within the crystal symmetry, such that the dimer is located with its axis of symmetry on an axis of twofold symmetry in the crystal. Therefore, only one monomer constitutes the asym- metric unit of this crystal form. In crystal form Iba2 o , however, the crystal symmetry takes no advantage of the internal symmetry. Hence, the asymmetric unit contains two dimers. Analytical ultracentrifugation showed that both human Iba proteins are able to dimerize to some extent. Nevertheless, the dimer formed in solution seems to differ from the crystallized Iba2 dimer because the ultracentrifugation experiments were con- ducted in the presence of a reducing agent and in the absence of Zn 2+ . Thus, disulfide bond formation, as well as Zn 2+ -assisted contacts were disfavored. The rather weak dissociation constants indicate that only a small fraction of the Iba proteins exists as dimers in vivo, although the dimerization process might be accelerated by other factors such as actin binding. The dimer in solution is probably analogous to the dimer observed in the m-Iba1 structure, where the dimeric form was obviously trapped in the crystallization process. Fig. 8. Iba2 (A–D) and Iba1 (E–H) are recruited into Shigella entry zones in an invasion assay. Iba2 is less concentrated in the cell periphery. HeLa cells, transiently transfected with CFP-Iba2 (C,L) or YFP-Iba1 (F,K), were infected with Shigella that were visualized as small rods by 4¢,6-diamidino-2- phenylindol DNA staining (D,H). F-actin was stained with Alexa-594–phallacidine (B,G). Overlay images (A,E) show more pro- nounced staining of membrane ruffle-like protrusions with Iba1. Double-transfected and infected cells confirm more pronounced recruitment into cellular protrusions of Iba1 (K) compared with Iba2 (L); overlay (I). J. O. Schulze et al. Human Iba proteins FEBS Journal 275 (2008) 4627–4640 ª 2008 The Authors Journal compilation ª 2008 FEBS 4633 The function of Iba proteins does not depend on Ca 2+ Equilibrium dialysis showed that neither Iba1 nor Iba2 bind Ca 2+ in presence of 100 lm Ca 2+ and ultracen- trifugation revealed no significant influence of Ca 2+ on the homodimerization. It is possible that the pro- teins bind calcium ions at higher concentrations than tested here. However, it should be noted that the Ca 2+ concentration found in the cytoplasm of mammalian cells, where the native Iba proteins are localized, is 0.1–10 lm. Calcium overlays demonstrated weak cal- cium binding of Iba1 EF-hand 1, but not of EF-hand 2 [3,25], in contrast to the m-Iba1 crystal structure [4], which revealed a calcium ion bound to EF-hand 2 only. Ca 2+ binding had been reported to be necessary for Iba1 function in membrane ruffling and phagocytosis [3] and to enhance the interaction of Iba1 with F-actin to a certain degree [11]. In this study F-actin binding and cross-linking by Iba proteins was calcium indepen- dent, confirming previous results for Iba1 [12,25]. These differing results may be due to the difficulties of quantifying actin binding exactly. Our study indicates that both Iba proteins neither bind nor depend on Ca 2+ for its function. We conclude that their actin binding and cross-linking activity has to be regulated by factors other than Ca 2+ . EF-hand 1 is functionally inactive Calcium ions in typical EF-hands are coordinated by six to seven oxygen atoms in pentagonal bipyramidal geometry. Classical EF-hand proteins like calmodulin and troponin C with a high Ca 2+ affinity possess three to four acidic residues that bind the calcium ion via their negatively charged side chains [18]. EF-hand 1 was not observed to bind Ca 2+ in any Iba structure. The conformation of the EF-hand 1 loop is very similar in all these structures: a type I b turn, which is stabilized by several hydrogen bonds. This b turn conformation is not observed in typical EF-hand loop structures [26]. It is very rare and has so far only been observed in the human S100 protein pso- riasin (S100A7) [27]. This loop conformation prevents binding of Ca 2+ to EF-hand 1 without prior spatial rearrangement. Although EF-hand 1 of the Iba pro- teins shows some resemblance with the consensus sequence, the crucial glutamate in the last position of the motif (the ‘)Z’ position) [28] is substituted mostly by serines or glycines. The coordination of Ca 2+ by the )Z glutamate is the prime reason for the move- ment of the outgoing helix and the conformational change of EF-hand proteins upon Ca 2+ binding [18]. Thus, it may be concluded that EF-hand 1 of the Iba proteins is not capable of functional Ca 2+ binding. Function of EF-hand 2 In both Iba1 crystal structures, the loops of EF-hand 2 adopt essentially the same conformation (Fig. 9C). In Iba2, however, residues 96–100 of the loop are in a dif- ferent, more open conformation, and a rearrangement would be necessary to bind a calcium ion. Closer inspection of both Iba1 crystal structures shows that the open loop conformation was not possible because of steric clashes with adjacent molecules in their crystal lattices. The NMR structure of m-Iba1 (PDB code Fig. 9. Superposition of Iba2 (blue) on (A) Ca 2+ -free h-Iba1 (dark green) [4] and (B) Ca 2+ -bound, dimeric m-Iba1 (light green) [4]. The second subunit of the m-Iba1 dimer is depicted in gray and calcium ions are shown as orange spheres. The relocation of helix aE, which is crucial for dimerization, is indi- cated by a black arrow. (C) Detailed view of EF-hand 2. The residues involved in Ca 2+ coordination of m-Iba1 are shown in stick representation. Human Iba proteins J. O. Schulze et al. 4634 FEBS Journal 275 (2008) 4627–4640 ª 2008 The Authors Journal compilation ª 2008 FEBS 2G2B) indicates that both conformations coexist in solution. Crystal structures with bound calcium ions could not be obtained for h-Iba1 [4] or for Iba2. Only in m-Iba1 was Ca 2+ observed to bind to EF-hand 2 [4]. It should be noted, however, that the Ca 2+ concentra- tion of 2.5 mm used for the crystallization is two orders of magnitude higher than that in the cytoplasm. This large discrepancy raises the question of whether the observed Ca 2+ binding can occur in vivo. In contrast to classical EF-hand proteins, the Ca 2+ in m-Iba1 is not coordinated in pentagonal bipyramid geometry, but resides in the center of a distorted tetra- hedron. There are additional contributions by the Thr100 carbonyl group at an unusual angle and a water molecule. A comparison of EF-hand 2 with the consensus motif [26] shows that it does not contain any acidic residues for the Ca 2+ coordination in the N-terminal half (Fig. 1). However, this first half of the loop is supposed to bind the calcium ion initially [18]. Furthermore, the last residue of the loop, the )Z posi- tion, is not a glutamate but a shorter aspartate residue, which cannot bind Ca 2+ in a bidentate manner as glutamate usually does. Overall, the Ca 2+ coordina- tion appears too weak to overcome the energy barrier of the conformational rearrangement giving rise to the dimerization observed in the m-Iba1 structure. More- over, the conformational change from the monomeric Iba structures to the dimeric m-Iba1 is very different from that of any known EF-hand protein. Surpris- ingly, it is not the outgoing helix aD of EF-hand 2, which shifts its location, but the incoming helix aC. Furthermore, there is a more pronounced rearrange- ment in EF-hand 1 than in EF-hand 2, although EF-hand 1 does not bind Ca 2+ . Therefore, it may be questioned if the rearrangement and dimerization of the Iba proteins are indeed caused by Ca 2+ binding. Cellular localization and recruitment to sites of Shigella invasion While intracellular localization patterns of Iba2 were similar to patterns seen with Iba1 constructs, Iba2 recruitment into peripheral structures was less pro- nounced, resulting in a less distinct peripheral pattern compared with Iba1 (Figs 7 and 8). We used a model for Shigella invasion of epithelial cells [22] to study Iba recruitment into bacteria-induced membrane ruffles. In this model, Rac and RhoA are recruited around enter- ing bacteria (peribacterial recruitment), whereas RhoC and the ERM protein ezrin accumulate in cellular protrusions [29]. Peribacterial protein recruitment is considered to be part of early invasion steps; whereas protein recruitment into membrane ruffles occurs at a later stage of infection [29]. In this study, no peribacte- rial recruitment of the Iba proteins was observed. In the Shigella invasion model of epithelial cells, CDC42, Rac and Rho are essential for efficient internalization of the bacteria [30–32]. Interestingly, Iba1 has been associated with Rac-mediated membrane ruffling in a variety of cells. However, direct protein–protein inter- action of Rac and Iba1 has not been reported [3,33,34]. Thus, our finding of Shigella-induced Iba recruitment into membrane ruffles, in contrast to the peribacterial staining pattern of Rac, is compatible with the view that Iba and Rac proteins do not directly interact with each other during Shigella inva- sion of epithelial cells. The peripheral staining pattern seen in the bacterial invasion model suggests a role for Iba downstream of Rac activation. This is in agree- ment with data showing inhibition by an Iba deletion mutant of cellular protrusions induced by constitu- tively active Rac [3]. A potential role for the Iba proteins in the generation and ⁄ or maintenance of Shigella-induced membrane ruffles is stabilizing mem- brane-associated actin filaments by cross-linking, simi- lar to Iba activity in phagocytes [12]. In addition to Iba, the actin cross-linking proteins a-actinin [32] and plastin [22] have been found in Shigella entry sites, showing recruitment patterns similar to Iba. Although the morphology of Iba-induced actin bundles has been described [12], it is not known whether Iba-mediated bundling of F-actin is sensitive to the orientation of actin filaments. Similarly, nothing is known about potentially functional differences between Iba isoforms due to the slightly varying recruitment patterns described here. Conserved surface residues and actin cross-linking In sequence alignments, highly conserved regions of the Iba proteins become apparent (Fig. 1). When the conservation of residues is plotted on the protein sur- face, Iba1 and Iba2 show very similar highly conserved regions (Fig. 10A,B). As expected, the residues com- prising the Iba1 dimerization interface are highly con- served in Iba1 as well as in Iba2. Surprisingly, the helix–loop–helix region aB–aC, which comprises the outgoing helix of EF-hand 1 and the incoming helix of EF-hand 2, is also strictly conserved; even though it is solvent exposed. This region contains several hydro- phobic residues on the surface and is almost uncharged, although it is surrounded by highly charged patches (Fig. 10C). This helix–loop–helix region undergoes a structural rearrangement upon J. O. Schulze et al. Human Iba proteins FEBS Journal 275 (2008) 4627–4640 ª 2008 The Authors Journal compilation ª 2008 FEBS 4635 dimerization in m-Iba1 [4] and was seen to be flexible in one of the four molecules in Iba2 o . Thus, this conserved region may constitute an interface for inter- action with another protein, possibly actin. It is likely that Iba monomers, due to their small size, have only one F-actin binding site. Consequently, F-actin cross-linking would require Iba homodimeriza- tion. We observed Iba homodimerization in our ana- lytical ultracentrifugation experiments and the crystal structure of mIba1 [4] shows a homodimer. The role of Iba homodimerization in F-actin cross-linking remains to be studied. Conclusion According to our studies, Iba1 and Iba2 share similar overall structures and molecular functions. They are able to cross-link actin, which probably requires dimerization of the Iba proteins. The actin cross-link- ing ability might play a role during the invasion of host cells by Shigella and other invasive pathogens. Although Iba2 generally appears to be less active than Iba1, the most outstanding difference between both Iba proteins seems to be their distinct expression patterns in various tissues of the body. Experimental procedures Cloning A full-length human Iba2 cDNA fragment (GenBank CAB66501) was amplified by PCR from the clone DKFZp761J191 [15]. Primers CTGGATCCTCGGGCGA GCTCAGCAAC and GACGGCGGCCGCTCAGGGCAG GCTAGCAATGTCT were used. The PCR product was cloned into the vector pQTEV (GenBank AY243506) using BamHI and NotI restriction sites and introduced into Escherichia coli SCS1 cells carrying the pRARE plasmid [35], and a resulting clone was used for overexpression of Iba2 (GenBank DQ000573, PSF ID 109968, RZPD ID PSFEp250B085). cDNA clones of the LIFEdb [16] for expression of human Iba2 as C-terminal YFP or N-terminal CFP fusion proteins were a gift from S. Bechtel and S. Wiemann (DKFZ Heidelberg). These plasmids contain the DKFZp761J191 ORF in the vectors pdEYFP-N1gen and pdECFP-C1amp, respectively [17]. The ORF of the human Iba1 cDNA clone IOH13810 (Invitrogen, Carlsbad, CA), corresponding to GenBank NM_001623, was obtained from the RZPD German Resource Center in the expression vector pDEST17-D18. BL21(DE3) E. coli cells were transformed for protein expression. The same cDNA was also obtained in the vectors pdEYFP-C1amp and pdEYFP-N1gen [17] for expression of C- and N-terminal YFP fusion proteins in mammalian cells. Fermentation and purification Iba2 was prepared for crystallization as follows. Clone ID 109968 was fermenter-grown to an D 600 of 8 in 4 L of SB medium (12 gÆL )1 bacto-tryptone, 24 gÆL )1 yeast extract, 0.4% v ⁄ v glycerol, 17 mm KH 2 PO 4 ,72mm K 2 HPO 4 ) supplemented with 20 lgÆmL )1 thiamine, 100 lgÆmL )1 ampicillin and 34 lgÆmL )1 chloramphenicol. Protein expres- sion was induced with 1 mm isopropyl thio- b -d-galactoside for 3 h at 37 °C. Cells were pelleted by centrifugation and washed with extraction buffer (20 mm Tris ⁄ HCl, pH 8.0, 300 mm NaCl, 0.5 mm EDTA, 1 mm phenylmethanesulfonyl fluoride, 5 mm 2-mercaptoethanol). Cells were lyzed, and cell lysates and proteins were stored at 4 °C. Protein Fig. 10. Conservation of surface residues illustrated on the m-Iba1 dimer [4] depicted in (A) cartoon and (B) surface representation (the molecule is rotated by 180° in comparison to Fig. 9). Considered are Iba1 sequences from 13 species (Homo sapiens, Macata mulatta, Mus musculus, Rattus norvegicus, Sus scrofa, Bos taurus, Ornithorhynchus anatinus, Suberites domuncula, Haliotis discus hannai, Cypri- nus carpio, Fugu rubripes, Pagrus major and Epinephelus awoara) and Iba2 sequences from four species (Homo sapiens, Pongo pygmaeus, Bos taurus and Mus musculus). Identical residues are colored in dark blue, moderately conserved residues in green and non-conserved residues in red. (C) Electrostatic potential on the surface of the m-Iba1 dimer. Human Iba proteins J. O. Schulze et al. 4636 FEBS Journal 275 (2008) 4627–4640 ª 2008 The Authors Journal compilation ª 2008 FEBS [...]... Iba2 t crystals belong to space group P3221 with cell con stants a = b = 70.5 and c = 95.2 A These crystals contain one Iba2 molecule per asymmetric unit with a VM value [36] of 4.0 A3ặDa)1 and a solvent content of 69% Iba2 o crystals belong to space group P21212 with cell con stants a = 71.5, b = 186.5 and c = 51.4 A A VM value Human Iba proteins of 2.5 A3ặDa)1 corresponding to a solvent content of. .. on poly(l-proline) Sepharose, enrichment of actin by a cycle of polymerization and depolymerization, isoform separation by hydroxyapatite chromatography, and a nal gel ltration step Actin cosedimentation and cross-linking assays Samples of actin (4 lm) were induced to polymerize by addition of 1 mm MgCl2 and 0.15 m KCl alone or in the presence of Iba1 or Iba2 , and incubated at room temperature for 23... Burlingame, CA) and imaged using a 100 ã FEBS Journal 275 (2008) 46274640 ê 2008 The Authors Journal compilation ê 2008 FEBS 4637 Human Iba proteins J O Schulze et al Fluoroplan oil immersion lens on a Zeiss Axioplan-2 microscope, and images were captured using a Zeiss Axiocam camera and axiovision imaging software In the present case, this value is a composite of the molecular mass values Mm and Md and the... gene iba1 in the major histocompatibility complex class III region encoding an EF hand protein expressed in a monocytic lineage Biochem Biophys Res Commun 224, 855862 2 Kawasaki H, Nakayama S & Kretsinger RH (1998) Classication and evolution of EF-hand proteins Biometals 11, 277295 3 Ohsawa K, Imai Y, Kanazawa H, Sasaki Y & Kohsaka S (2000) Involvement of Iba1 in membrane rufing and phagocytosis of macrophages... 7.4, 50 mm NaCl, 0.02% NaN3, 2 mm dithiothreitol and 0.1 mm EDTA) were added to 400 lL of reservoir solution (1315% w v PEG 4000, 100 mm Na-acetate, pH 5.0 and 100 mm zinc acetate) Crystals of two distinct morphologies grew within 2 weeks: an orthorhombic crystal form (Iba2 o) with a size of 1000 ã 300 ã 50 lm and trigonal crystals (Iba2 t) with sizes of 500 ã 400 ã 400 lm Prior to X-ray data collection,... Sasaki Y & Kohsaka S (2004) Microglia macrophage-specic protein Iba1 binds to mbrin and enhances its actin-bundling activity J Neurochem 88, 844856 14 Bussow K, Scheich C, Sievert V, Harttig U, Schultz J, ă Simon B, Bork P, Lehrach H & Heinemann U (2005) Structural genomics of human proteins target selection and generation of a public catalog of expression clones Microb Cell Fact 4, 21 15 Wiemann S, Weil... Bauersachs ă ă S, Blum H et al (2001) Toward a catalog of human genes and proteins: sequencing and analysis of 500 novel complete protein coding human cDNAs Genome Res 11, 422435 16 Bannasch D, Mehrle A, Glatting KH, Pepperkok R, Poustka A & Wiemann S (2004) LIFEdb: a database for functional genomics experiments integrating information from external sources, and serving as a sample tracking system Nucleic... 36 Matthews BW (1968) Solvent content of protein crystals J Mol Biol 33, 491497 37 Heinemann U, Bussow K, Mueller U & Umbach P ă (2003) Facilities and methods for the high-throughput crystal structural analysis of human proteins Acc Chem Res 36, 157163 38 Kabsch W (1993) Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants J Appl Crystallogr... Allograft inammatory factory-1 A cytokine-responsive macrophage molecule expressed in transplanted human hearts Transplantation 61, 13871392 Human Iba proteins 9 Autieri MV, Carbone C & Mu A (2000) Expression of allograft inammatory factor-1 is a marker of activated human vascular smooth muscle cells and arterial injury Arterioscler Thromb Vasc Biol 20, 17371744 10 Kohler C (2007) Allograft inammatory... building and structural analysis [43] The structures were validated using what if [44] and procheck [45] Molecular drawings were prepared using pymol [46] lsqkab of the CCP4 suite [39] was used to calculate rmsd values and dali [47] to identify structural protein homologs Purication of actin Non-muscle b actin was puried from bovine brain [48,49] Briey, the method involved afnity purication of prolinactin . efficiency of Iba1 and Iba2 or in the overall morphology of the generated filament bundles. Calcium affinity of Iba1 and Iba2 Homodimerization and actin binding of. here reveals functional simi- larities and differences between Iba1 and Iba2 . We investigated Ca 2+ binding and homodimerization of Iba1 and Iba2 . Furthermore,